Hypertension and diabetes are the principal cause for chronic kidney disease (CKD). In both diseases, a high percentage of patients show activation of the renin angiotensin system (RAS). Renin is the rate-limiting enzyme in the activation of the RAS. Thus, understanding the mechanism and proteins involved in the release of renin may offer alternative targets for hypertension and CKD. Renin is stored in dense-core granules in juxtaglomerular (JG) cells, located at the pole of the renal afferent arteriole, in the kidney cortx. In hypertension and diabetes, reactive oxygen species, including hydrogen peroxide (H2O2), are enhanced in the kidney cortex. We found that hydrogen peroxide stimulate renin release from JG cells. However, the enzymes responsible for production of hydrogen peroxide in JG cells have not been identified. Hydrogen peroxide is mainly a product of superoxide dismutation or enzymatic formation by NADPH oxidases (NOX1-5). The NOX4 isoform preferentially produces hydrogen peroxide and its expression in the renal cortex is enhanced in diabetes and hypertension. Our preliminary data shows that NOX4 is expressed in renin granules in JG cells, suggestive of intragranular production of H2O2. However it is not known whether NOX4 produces the pool of hydrogen peroxide that stimulates renin release from JG cells; and whether NOX4/H2O2-induced renin release contributes to increase blood pressure and kidney damage in diabetic nephropathy. In addition the mechanism by which H2O2 stimulate renin release is unknown. In other cells H2O2 induces signaling by oxidation of protein thiols. In this proposal we will test the hypothesis that the NADPH oxidase isoform NOX4 produces hydrogen peroxide in juxtaglomerular cells and stimulate renin release, thereby increasing blood pressure and contributing to glomerular damage. We will also explore the protein targets by which H2O2 stimulates renin release. In Aim 1 we will use primary cultures of juxtaglomerular cells, isolated afferent arterioles and Akita mice to test the role of NOX4-derived hydrogen peroxide in renin release in vitro and in vivo. In Aim 2 we will use a proteomics approach and subcellular fractionation of renin granules, to identify proteins that are oxidized by hydrogen peroxide in juxtaglomerular cells. This approach will allow us to collect critical preliminary data for an RO1 submission and focus on new protein targets of hydrogen peroxide that mediate renin release.